The chemoselective ligation of peptides can be used to effect the total chemical synthesis of proteins.1 The most common ligation method, native chemical ligation, relies on the presence of a cysteine residue at the N-terminus of each ligation junction.2,3 Recently, we have reported4 a peptide ligation method, a “Staudinger Ligation,” that is universal, i.e., independent of the presence of any particular side chain This method is based on the Staudinger reaction, wherein a phosphine reduces an azide via a stable iminophosphorane intermediate.5 Acylation of this iminophosphorane yields an amide.6,7 
Scheme 1 illustrates our Staudinger Ligation which is further described in PCT application PCT/01/15440, filed May 11, 2000. A peptide fragment having a C-terminal phosphinothioester (2) reacts with another peptide fragment having an N-terminal azide (3). The resulting iminophosphorane (4) leads, after an S- to N-acyl shift, to an amidophosphonium salt (5). The P—N bond of the amidophosphonium salt is hydrolyzed readily to produce the amide product (6) and a phosphine oxide (7). Importantly, no residual atoms remain in the amide product.4,6b, so the ligation is traceless. The phosphinothioester (2) is prepared by reaction of a of phosphinothiol reagent (1), such as Ph2CH2—SH, where Ph is a phenyl group. The Staudinger Ligation can generally be employed to form peptide bonds and as such can be employed to ligate two amino acids, a peptide or a protein with an amino acid or peptide or two proteins. More generally, the Staudinger Ligation can be employed to form amide bonds. The amide bond is formed between a thioester and an azide. In general, the reaction functions for any thioester and any azide. The thioester is converted into a phosphinothioester which then reacts with the azide. For example, the thioester group may be formed, at the carboxy group of an amino acid or at the carboxy terminus of a peptide or protein or at an acid side group of an amino acid or one or more amino acids in a peptide or protein. The azido group may be formed, for example, at the amino group of an amino acid or at the amino terminus of a peptide or protein or at a basic side group of an amino acid or one or more amino acids in a peptide or protein. The Staudinger Ligation may also be employed to ligate an amino acid, peptide or protein group to a carbohydrate group, which may be a mono-, di-, tri- or polysaccharide, or to a nucleoside. The Staudinger Ligation may also be employed to ligate an amino acid, a peptide or protein group to a reporter group, tag or label (e.g., a group whose presence can be detected by optical or mass spectrometry or other instrumental method), including a fluorescent or phosphorescent group, an isotopic label or a radiolabel.
All natural α-amino acids except glycine have a stereogenic center at their α-carbon.8 To be an effective tool for the total chemical synthesis of proteins, a peptide ligation reaction must proceed without epimerization. The coupling of thioesters in native chemical ligation, which like the Staudinger Ligation (Scheme 1) involves transthioesterification followed by an S- to N-acyl shift,2,3 is known to proceed without detectable racemization.9 We have demonstrated that the Staudinger Ligation (Scheme 1) proceeds in near quantitative yield without detectable epimerization.
The Staudinger Ligation of Scheme 1 employs a phosphinothiol reagent (1). Previously reported methods of synthesis of such reagents4a, 4b generally proceed in low yield. Synthesis of the phosphinothiol of formula 1 where R and R′ are phenyl groups requires four synthetic steps, two of which are problematic, with an overall yield of about 39%. Difficulties can also be encountered in the synthesis of reagents of formula 1 where R and R′ are small alkyl groups, such as ethyl groups, due to instability of the reagent itself. Use of the Staudinger Ligation for the formation of amide bonds between a variety of species would be facilitated by the development of improved methods for the synthesis of phosphinothiol reagents and the development of such reagents with increased stability. This invention provides improvements for carrying out the Staudinger Ligation.

